Cell cycle regulators control mesoderm specification in human pluripotent stem cells

The mesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys, and the cardiovascular system originate. Understanding the mechanisms that control mesoderm specification could inform many applications, including the development of regenerative medicine therapies to manage diseases affecting these tissues. Here, we used human pluripotent stem cells to investigate the role of cell cycle in mesoderm formation. To this end, using small molecules or conditional gene knockdown, we inhibited proteins controlling G₁ and G₂/M cell cycle phases during the differentiation of human pluripotent stem cells into lateral plate, cardiac, and presomitic mesoderm. These loss-of-function experiments revealed that regulators of the G₁ phase, such as cyclin-dependent kinases and pRb (retinoblastoma protein), are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations disclosed that inhibition of the G₂/M regulator cyclin-dependent kinase 1 decreases BMP (bone morphogenetic protein) signaling activity specifically during lateral plate mesoderm formation while reducing fibroblast growth factor/extracellular signaling-regulated kinase 1/2 activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.